SKP2 siRNA inhibits the degradation of P27kip1 and down-regulates the expression of MRP in HL-60/A cells.
SKP2 siRNA inhibits the degradation of P27kip1 and down-regulates the expression of MRP in HL-60/A cells.
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SKP2 siRNA 抑制 P27kip1 的降解并下调 HL-60/A 细胞中 MRP 的表达。
DOI：
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发表时间：
2009
影响因子：
3.7
通讯作者：
Jian
中科院分区：
文献类型：
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作者：
Jie Xiao;S. Yin;Yiqing Li;Shuangfeng Xie;D. Nie;Li;Xiuju Wang;Yu;Jian
S相激酶相关蛋白2(Skp2)基因是一种肿瘤抑制基因，参与泛素介导的P27kip1的降解。Skp2和P27kip1通过调节白血病细胞的增殖、凋亡和分化，影响白血病的进展和预后。本研究探讨了Skp2下调逆转HL-60/A耐药的机制。用带有Skp2 siRNA的Amaxa核受体系统对HL-60/A细胞进行核转录。逆转录-聚合酶链式反应检测Skp2、P27kip1和多药耐药相关蛋白(MRP)的基因和蛋白表达水平；流式细胞仪分析细胞周期。根据两种细胞在不同浓度化疗药物作用下的死亡率进行细胞毒性分析，计算其半数抑制浓度，比较两种细胞对化疗药物的敏感性。HL-60/A细胞对阿霉素、柔红霉素和阿糖胞嘧啶的交叉耐药表现为多药耐药表型特征，这是由于MRP的表达。我们发现在HL-60/A细胞中Skp2的表达高于HL-60细胞，而P27kip1的表达低于HL-60细胞。Skp2 siRNA作用于HL-60/A细胞后，Skp2蛋白表达下调，而P27kip1蛋白表达上调。与对照组相比，Skp2 siRNA核修饰的HL-60/A细胞MRP的mRNA和蛋白表达水平较低，而后者对阿霉素、柔红霉素和阿糖胞苷的敏感性较高。下调Skp2表达，将细胞阻滞在G0/G1期，可改善MRP表达下调的白血病细胞对药物的敏感性。
S-phase kinase-associated protein 2 (SKP2) gene is a tumor suppressor gene, and is involved in the ubiquitin-mediated degradation of P27kip1. SKP2 and P27kip1 affect the proceeding and prognosis of leukemia through regulating the proliferation, apoptosis and differentiation of leukemia cells. In this study, we explored the mechanism of reversing of HL-60/A drug resistance through SKP2 down-regulation. HL-60/A cells were nucleofected by Amaxa Nucleofector System with SKP2 siRNA. The gene and protein expression levels of Skp2, P27kip1, and multi-drug resistance associated protein (MRP) were determined by reverse transcription-polymerase chain reaction and western blot analysis, respectively. The cell cycle was analyzed by flow cytometry. The 50% inhibitory concentration value was calculated using cytotoxic analysis according to the death rate of these two kinds of cells under different concentrations of chemotherapeutics to compare the sensitivity of the cells. HL-60/A cells showed multi-drug resistance phenotype characteristic by cross-resistance to adriamycin, daunorubicin, and arabinosylcytosine, due to the expression of MRP. We found that the expression of SKP2 was higher in HL-60/A cells than in HL-60 cells, but the expression of P27kip1 was lower. The expression of SKP2 in HL-60/A cells nucleofected by SKP2 siRNA was down-regulated whereas the protein level of P27kip1 was up-regulated. Compared with the MRP expression level in the control group (nucleofected by control siRNA), the mRNA and protein expression levels of MRP in HL-60/A cells nucleofected by SKP2 siRNA were lower, and the latter cells were more sensitive to adriamycin, daunorubicin, and arabinosylcytosine. Down-regulating the SKP2 expression and arresting cells in the G0/G1 phase improve drug sensitivity of leukemia cells with down-regulated MRP expression.
影响因子：
20.3
作者：
Barata, JT;Cardoso, AA;Boussiotis, VA
通讯作者：
Boussiotis, VA
影响因子：
20.3
作者：
Agarwal, Anupriya;Bumm, Thomas G. P.;Deininger, Michael W.
通讯作者：
Deininger, Michael W.